Abstract
Although docosahexaenoic acid (DHA), an important dietary omega-3 polyunsaturated fatty acid (PUFA), is at present primarily sourced from marine fish, bioengineered crops producing DHA may offer a more sustainable and cost-effective source. DHA has been produced in transgenic oilseed crops, however, DHA in seed oil primarily occupies the sn-1/3 positions of triacylglycerol (TAG) with relatively low amounts of DHA in the sn-2 position. To increase the amount of DHA in the sn-2 position of TAG and in seed oil, putative lysophosphatidic acid acyltransferases (LPAATs) were identified and characterized from the DHA-producing alga Schizochytrium sp. and from soybean (Glycine max). The affinity-purified proteins were confirmed to have LPAAT activity. Expression of the Schizochytrium or soybean LPAATs in DHA-producing Arabidopsis expressing the Schizochytrium PUFA synthase system significantly increased the total amount of DHA in seed oil. A novel sensitive band-selective heteronuclear single quantum coherence (HSQC) NMR method was developed to quantify DHA at the sn-2 position of glycerolipids. More than two-fold increases in sn-2 DHA were observed for Arabidopsis lines expressing Schizochytrium or soybean LPAATs, with one Schizochytrium LPAAT driving DHA accumulation in the sn-2 position to 61% of the total DHA. Furthermore, expression of a soybean LPAAT led to a redistribution of DHA-containing TAG species, with two new TAG species identified. Our results demonstrate that transgenic expression of Schizochytrium or soybean LPAATs can increase the proportion of DHA at the sn-2 position of TAG and the total amount of DHA in the seed oil of a DHA-accumulating oilseed plant. Additionally, the band-selective HSQC NMR method that we developed provides a sensitive and robust method for determining the regiochemistry of DHA in glycerolipids. These findings will benefit the advancement of sustainable sources of DHA via transgenic crops such as canola and soybean.
Highlights
Docosahexaenoic acid (DHA) is an important dietary omega-3 long-chain polyunsaturated fatty acid (LC-PUFA) for human health
Sn-2 DHA could not be detected in TAG from DHA-accumulating Arabidopsis lines containing 1.4% (T2 line) and 2.9% total DHA (T3 line), and a T2 canola line containing 1.2% total DHA (S1B Fig)
The two novel DHA-containing TAG species identified were TAG 60:8+NH4 and TAG 60:10+NH4. By searching this subset of DHA-TAG species with additional acyl-fragment-ion masses, we predict the three acyl chains to be 20:1, 22:6, & 18:1 for TAG 60:8+NH4 (976.8 m/z) and 20:1, 22:6, & 18:3 for TAG 60:10+NH4 (972.8 m/z). These results show that heterologous expression of a soybean lysophosphatidic acid acyltransferases (LPAATs) in Arabidopsis alters the TAG composition in seed oil and provides further evidence that these LPAATs have in planta selectivity for DHA-CoA substrate
Summary
Docosahexaenoic acid (DHA) is an important dietary omega-3 long-chain polyunsaturated fatty acid (LC-PUFA) for human health. There is increasing demand for DHA that is typically sourced from marine fish. Fish accumulate DHA via the food chain from marine algae primary producers. These algae can synthesize DHA either by an oxygen-dependent fatty acid desaturation and elongation pathway that is widespread in nature or via a unique anaerobic polyunsaturated fatty acid (PUFA) synthase system that has similarities to microbial polyketide synthases [3]. Alternative sustainable and cost-effective sources of DHA by producing omega-3 LC-PUFAs in oilseed crops are of great commercial interest [8]
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